Microbial fermentation and biotransformation reactions are being employed with increasing frequency in the production of a number of commercially and industrially important products. There is also growing interest in developing alternative energy sources through microbial fermentation of waste materials. The economic feasibility of these processes depends on maximizing the efficiency of the fermentation or biotransformation reactions.
Bacterial species are able to use various energy sources, including light and diverse organic and inorganic chemicals, for growth and metabolism. These energy sources are used to produce an electrochemical gradient that provides an electron donor for metabolism and allows maintenance of a membrane potential and proton motive force. The energetics of living systems are driven by electron transfer processes in which electrons are transferred from a substrate, which is thereby oxidized, to a final electron acceptor, which is thereby reduced.
In microbial metabolism, the energy produced from the driving force of electrons is directly proportional to the potential energy difference (xcex94E0xe2x80x2) between the initial electron donor (the first biochemical dehydrogenating reaction) and final electron acceptor (e.g., the final biochemical hydrogenating reaction).
Certain microorganisms (e.g., Escherichia and Actinobacillus) are able to grow using H2 as an electron donor to reduce fumarate into succinate in an anaerobic respiration process. These bacteria obtain free energy and reducing power from the electron driving force generated by the E0xe2x80x2 difference between the coupled oxidoreduction half reactions of [2H+/H2] and [fumarate/succinate].
Methanogens are strict anaerobic archea that can couple H2 or HCOOH oxidation to CO2 reduction into methane. Methanogenesis produces less free energy than other anaerobic respiration processes (e.g., fumarate, nitrate, or sulfate reduction) because the E0xe2x80x2 difference between the half oxidation reduction reactions of [2H+/H2] and [CO2/CH4] is relatively small.
Hydrogen oxidation by microbial hydrogenases can be coupled to reduction of various biological electron carriers including NAD+, cytochromes, and quinones or to certain artificial redox dyes, such as methyl-viologen and neutral red (NR) (Annous, et al., 1996, Appl. Microbiol. Biotechnol. 45:804-810, Kim, et al., 1992, J. Microbiol. Biotechnol. 2:248-254). The effect of redox dyes, with or without electrochemical reduction systems, on metabolite patterns and H2 production has been examined in several microbial processes, including the glutamate (Hongo, et al., 1979, Agric. Biol. Chem. 43:2083-2986), butanol (Girbal, et al., 1995, Microbiol. Rev. 16:151-162 and Kim, et al., 1992, J. Microbiol. Biotechnol, 2:268-272), and butyrate (Shen, et al., 1996, Appl. Microbiol, Biotechnol, 45:355-362) fermentations.
The specific activities of redox enzymes involved in bacterial catabolism, such as hydrogenase or fumarate reductase, can be measured using their in vivo electron carriers (e.g., NAD or menanquinone) or with artificial redox dyes (e.g., benzyl viologen) (Cecchini, et al., 1986, Proc. Natl. Acad. Sci. USA 83:8898-8902, Dickie, et al., 1979, Can. J. Biochem., 57:813-821, Kemner, et al., 1994, Arch. Microbiol., 161:47-54, Petrov, et al., 1989, Arch. Biochem. Bio-phys. 268:306-313, and Wissenbach, et al., 1990, Arch. Microbiol. 154:60-66). Bacteria that produce succinic acid as a major catabolic end product (e.g., E. coli, Wolinella succinogenes and other species) have a fumarate reductase (FRD) complex that catalyzes fumarate-dependent oxidation of menaquinone. This reaction is coupled to the generation of a transmembrane proton gradient that is used by the organism to support growth and metabolic function (Kortner, et al., 1992, Mol. Microbiol. 4:855-860 and Wissenbach, et al., 1992, Arch. Microbiol. 158:68-73). The fumarate reductase of E. coli is composed of four nonidentical subunits: FRDA, FRDB, FRDC, and FRDD. The subunits are arranged in two domains: (i) the FRDAB catalytic domain and the FRDCD membrane anchor domain, which is essential for electron transfer and proton translocation reactions involving menaquinone (Cecchini, et al., 1995, J. Bacteriol. 177:4587-4592, Dickie, et al., 1979, Can. J. Biochem., 57:813-821, and Westenberg, et al., 1990, J. Biol. Chem. 265:19560-19567). Subunits FRDA and FRDB retain catalytic activity in solubilized membrane preparations.
Electrochemical techniques employing redox dyes are useful for investigating the oxidation-reduction characteristics of biological systems and provide information about biological energy metabolism (Moreno, et al., 1993, Eur. J. Biochem. 212:79-86 and Sucheta, et al., 1993, Biochemistry 32:5455-5465). Redox dyes that are useful in bioelectrochemical systems must easily react with both the electrode and the biological electron carriers. Many biological electron carriers, such as NAD (Miyawaki, et al., 1992, Enzyme Microb. Technol. 14:474-478 and Surya, et al., 1994, Bioelectrochem. Bioenerg. 33:71-73), c-type cytochromes (Xie, et al., 1992, Bioelectrochem. Bioenerg. 29:71-79), quinones (Sanchez, et al., 1995, Bioelectrochem. Bioenerg. 36:67-71), and redox enzymes, such as nitrite reductase (White, et al., 1987, Bioelectro-chem. Bioenerg. 26:173-179), nitrate reductase (Willner, et al., 1992, Bioelectrochem. Bioenerg. 29:29-45), fumarate reductase (Sucheta, et al., 1993, Biochemistry. 32:5455-5465), glucose-6-phosphate dehydrogenase (Miyawaki, et al., 1992, Enzyme Microb. Technol. 14:474-478), ferredoxin-NADP reductase (Kim, et al., 1992, J. Microbiol. Biotechnol. 2:2771-2776) and hydrogenase (Schlereth, et al., 1992, Bioelectrochem. Bioenerg. 28:473-482) react electrochemically with the redox dyes.
Certain redox dyes with lower redox potentials than that of NAD, such as methyl viologen (MV) (Kim, et al., 1988, Biotechnol. Lett. 10:123-128, Pequin, et al., 1994, Biotechnol. Lett. 16:269-274, and White, et al., 1987, FEMS Microbiol. Lett. 43:173-176), benzyl viologen (Emde, et al., 1990, Appl. Environ. Microbiol. 56:2771-2776), and neutral red (NR) (Girbal, et al., 1995, FEMS Microbiol. Rev. 16:151-162 and Kim, et al., J. Biotechnol. 59:213-220) have been correlated with alterations in the rate of biological redox reactions in vivo. Hongo and Iwahara (Hongo, et al., 1979, Agric. Biol. Chem. 43A:2075-2081 and Hongo, et al., 1979, Agric. Biol. Chem. 43B:2083-2086) discovered that including redox dyes with low xcex94E0xe2x80x2 values (e.g., MV, benzyl viologen and NR) in bacterial fermentation conducted under cathodic reduction conditions was correlated with an increase in L-glutamate yield (about 6%). In the method of Hongo and Iwahara, a platinum electrode was used to deliver electricity at a level that was sufficiently high to generate hydrogen from water. Therefore, the source of increased reducing power in the method of Hongo and Iwahara is not known, nor was the mechanism by which the tested dyes affect fermentation characterized. Addition of NR to acetone-butanol fermentations is correlated with decreased production of acids and H2, and enhanced production of solvent (Girbal, et al., 1995, FEMS Microbiol. Rev. 16:151-162 and Kim, et al., 1992, J. Microbiol. Biotechnol. 2:2771-2776), an effect that was further enhanced under electroenergized fermentation conditions (Ghosh, et al., 1987, abstr. 79. In Abstracts of Papers, 194th ACS National Meeting. American Chemical Society). Viologen dyes have been used as electron mediators for many electrochemical catalytic systems using oxidoreductases in vitro and in vivo (James, et al., 1988, Electrochem. Bioenerg. 20:21-32, Kim, et al., 1988, Biotechnol. Lett. 10:123-128, Moreno, et al., 1993, Eur. J. Biochem. 212:79-86, Schlereth, et al., 1992, Bioelectrochem. Bioenerg. 28:473-482, and White, et al., 1987, FEMS Microbiol. Lett. 43:173-173).
An electrochemical system was used to regenerate reduced iron for growth of Thiobacillus ferrooxidans on electrical reducing power (Robinson, et al., 1982, Can. J. Biochem. 60:811-816).
It may be possible to control or alter metabolism by linking biochemical processes to an external electrochemical system. Linking biochemical and electrochemical systems may allow the use of electricity as a source of electrons for bacterial growth and in vivo or in vitro fermentation or biotransformation reactions.
A reversible biochemical-electrochemical link may allow conversion of microbial metabolic or enzyme catalytic energy into electricity. Biofuel cells in which microbial energy is directly converted to electrical energy using conventional electrochemical technology have been described (Roller, et al., 1984, J. Chem. Tech. Biotechnol. 34B:3-12 and Allen, et al., 1993, Appl. Biochem. Biotechnol. 39-40:27-40). Chemical energy can be converted to electric energy by coupling the biocatalytic oxidation of organic or inorganic compounds to the chemical reduction of the oxidant at the interface between the anode and cathode (Willner, et al., 1998, Bioelectrochem. Bioenerg. 44:209-214). However, direct electron transfer from microbial cells to electrodes has been shown to take place only at very low efficiency (Allen, et al., 1972, J. R. Norris and D. W. Ribbons (eds.), Academic Press, New York, 6B:247-283).
The electron transfer efficiency can be improved by using suitable redox mediators (Bennetto, et al., 1985, Biotechnol. Lett. 7:699-105), and most of the microbial fuel cells studied employed electron mediators such as the redox dye thionin (Thurston, et al., 1985, J. Gen. Microbiol. 131:1393-1401). In microbial fuel cells, two redox couples are required for: (1) coupling the reduction of an electron mediator to bacterial oxidative metabolism; and (2) coupling the oxidation of the electron mediator to the reduction of the electron acceptor on the cathode surface (where the electron acceptor is regenerated by atmospheric oxygen) (Ardeleanu, et al., 1983, Bioelectrochem. Bioenerg. 11:273-277 and Dealney, et al., 1984, Chem. Tech. Biotechnol. 34B:13-27).
The free energy produced by either normal microbial metabolism or by microbial fuel cell systems is mainly determined by the potential difference (xcex94E0xe2x80x2) between the electron donor and acceptor according to the equation, xe2x88x92xcex94G=nFxcex94E0 in which xcex94G is the variation in free energy, n is the number of electron moles, and F is the Faraday constant (96,487 J/volt) (Dealney, et al., 1984, Chem. Tech. Biotechnol. 34B:13-27). Coupling of the metabolic oxidation of the primary electron donor (NADH) to the reduction of the final electron acceptor (such as oxygen or fumarate in bacterial respiration systems) is very similar to the coupling of electrochemical half-reaction of the reductant (electron donor) to the half reaction of the oxidant (electron acceptor) in a fuel cell or battery system (Chang, et al., 1981, 2nd ed., Macmillan Publishing, New York). Biological reducing power sources such as NADH (E0xe2x80x2=xe2x88x920.32 volt), FdH2 (E0xe2x80x2=xe2x88x920.42 volt), or FADH2 (E0xe2x80x2=xe2x88x920.19 volt) with low redox potentials can act as reductants for fuel cells, but they are not easily converted to electricity because the cytoplasmic membrane must be non-conductive to maintain the membrane potential absolutely required for free energy (i.e., ATP) production (Thauer, et al., 1997, Bacteriol. Rev. 41:100-180).
For electron transfer to occur from a microbial electron carrier to an electrode, an electron mediator is required (Fultz, et al., 1982, Anal. Chim. Acta. 140:1-18). Allen, et al. (1993, Appl. Biochem. Biotechnol. 39-40:27-40) reported that the reducing power metabolically produced by Proteus vulgaris or E. coli can be converted to electricity by using electron mediators such as thionin. Tanaka, et al. (1985, Chem. Tech. Biotechnol. 35B:191-197 and 1988, Chem. Tech. Biotechnol. 42:235-240) reported that light energy can be converted to electricity by Anabaena variabilis using HNQ as the electron mediator. Park, et al. (1997, Biotech. Techniq. 11:145-148) confirmed that viologen dye cross-linked with carbon polymers and adsorbed to Desulfovibro desulfuricans cytoplasmic membranes can mediate electron transfer from bacterial cells to electrodes or from electrodes to bacterial cells.
There remains a need in the art for improved, more efficient methods for converting metabolic reducing power to electrical energy, and for converting electrical energy to metabolic reducing power.
One aspect of the present invention is a method of promoting reductive processes in a bioreactor system comprising the steps of (a) providing an electrochemical bioreactor system having a cathode compartment equipped with a cathode and an anode compartment equipped with an anode, the cathode and anode compartment being separated by a cation selective membrane, wherein the cathode and anode are connected by a conductive material to a power supply; (b) placing a suitable amount of neutral red and a biological catalyst in the cathode compartment.
In a particularly advantageous form of the invention, the biological catalyst is an enzyme that uses NADH or NADPH as a cofactor. The cathode compartment comprises NADH or NADPH and an oxidized substrate for the enzyme. Electrically reduced neutral red transfers electrons to NAD30  or NADP30 . In a preferred form of the invention, the enzyme is oxidoreductase, most particularly in alcohol dehydrogenase, the oxidized substrate is an aldehyde or ketone and the reduced product is an alcohol.
Another aspect of the invention is a method for generating electricity using a biological system comprising the steps of (a) providing an electrochemical fuel cell system comprising an anode compartment and a cathode compartment separated by a cation-selective membrane, wherein each compartment is equipped with an electrode, wherein the electrodes are connected by a wire to a multimeter; (b) placing an anolyte in the anode compartment, the anolyte comprising a suitable concentration of neutral red and a biological catalyst selected from the group consisting of bacteria, archea, plant cells, and animal cells; (c) placing a suitable catholyte in the cathode compartment; and (d) allowing the neutral red-mediated conversion of chemical reducing power to electricity.
It is an object of the invention to provide methods that allow the interconversion of biochemical reducing power (e.g., NADH), biological energy (ATP), and electrical energy in an electrochemical bioreactor or fuel cell.
It is a further object of the invention to provide an economical method of promoting cell growth or production of desired products using electrically reduced neutral red.
Another object of the invention is to provide a method for converting biological reducing power into electricity.
It is an advantage of the present invention that electrical energy may be used to promote cell growth or fermentation or enzymatic transformation in the presence of neutral red.
Another advantage of the invention is that neutral red promotes the generation of electrical energy from waste material comprising mixed bacterial populations.
Other objects, features, and advantages of the present invention will be apparent on review of the specification and claims.